X-RAY CRYSTALLOGRAPHIC STRUCTURE OF RECOMBINANT EOSINOPHIL-DERIVED NEUROTOXIN AT 1.83-ANGSTROM RESOLUTION

The X-ray crystallographic structure of recombinant eosinophil-derived neurotoxin (rEDN) has been determined by molecular replacement method s and refined at 1.83 Angstrom resolution to a conventional R-factor ( =Sigma parallel to F-o\-\F-c parallel to/Sigma\F-o\) of 0.152 with exc ellent stereochemistry. The molecular model of rEDN contains all 1081 non-hydrogen protein atoms, two non-covalently bound sulfate anions an d 121 ordered solvent molecules. The polypeptide fold of rEDN is relat ed to those observed in the homologous structures of RNase A, Onconase and angiogenin. rEDN is one of the largest members of the pyrimidine- specific ribonuclease superfamily of vertebrates and has small inserti ons in four of its seven loop structures and a large insertion from As p115 to Tyr123. The non-covalently bound SO4(A) and SO4(B) anions occu py phosphate-binding subsites of rEDN. The active site SO4(A) anion ma kes contacts in rEDN that are similar to those in RNase A and involve the side-chain atoms of Gln14, His15 and His129, and the NH group of L eu130. The SO4(B) anion makes contacts with the side-chain atoms of Ar g36 and Asn39 and the main-chain atoms of; Asn39 and Gln40. The equiva lent residues of RNase A cannot make contacts similar to those observe d in rEDN, The SO4(B) binding site of rEDN likely corresponds to the P --1 subsite and may be representative of how other homologous RNases b ind the P--1 phosphate. (C) 1996 Academic Press Limited